Progesterone and its metabolite 5α-dihydroprogesterone (5α-DHP) have been shown to bring about gonadotropin release in the estrogen-primed ovariectomized rat. One of the actions of progesterone is the decrease in occupied estrogen receptors (E2RS) in the anterior pituitary and uterus. This study attempted to determine if 5α-DHP had a similar effect on pituitary and uterine E2RS. Estrogen-primed ovariectomized mature female rats were injected with either vehicle, or 0.2, 0.8 or 2 mg/kg body weight (BW) of 5α-DHP, 2 h before sacrifice and 1 h before the last estradiol injection (2 µg/rat). Nuclear E2RS were determined by Scatchard analyses in the uterus and anterior pituitary. Total nuclear E2R levels of both tissues showed a 2-fold increase in the number of estradiol-binding sites after estradiol administration, as compared to control groups. In estradiol-primed rats, 5α-DHP produced a significant decrease in total nuclear E2R levels in a tissue-specific manner. In the pituitary, there was a maximal and significant decrease in nuclear E2RS with 0.2 and 2.0 mg/kg BW of 5α-DHP as compared to estradiol alone; the intermediate dose of 0.8 mg/kg BW of 5α-DHP induced a smaller nonsignificant change in nuclear E2RS. In the uterus, 5α-DHP showed a dose-dependent decrease in nuclear E2RS. The 5α-DHP effect in both tissues was due to a specific reduction in the occupied form of nuclear E2RS levels. The unoccupied form of E2RS was unaffected by 5α-DHP administration. 5α-DHP did not have any effect in the absence of estrogen priming. The acute tissue-specific decrease in occupied E2RS induced by 5α-DHP was similar to that previously described by us with progesterone in the same animal model. The dose of 5α-DHP required was less than the dose of progesterone for compa